Phosphatase inhibitor and protease inhibitor cocktails

M‐PER (Thermo Fisher Scientific, Waltham, Massachusetts, USA) with phosphatase inhibitor and protease inhibitor cocktails (Roche, Basel, Switzerland) was applied to lyse the cells, and then target verification by drug affinity responsive target stability (DARTS) was performed.¹², ¹³ The cell lysates were added to TNC buffer (10 mM CaCl2, 50 mM NaCl and 50 mM Tris‐HCl pH 8.0) and incubated with either 4‐IPP or the vehicle (DMSO) on ice for 1 h and then at room temperature for another 20 min. Then, the cell lysates were digested at room temperature for 20 min by Pronase (Roche, Basel, Switzerland), which was stopped using SDS loading buffer, after which a heat treatment was applied at 70 °C for 10 min. SDS‐PAGE and western blotting were then performed for the indicated proteins.

A total 1 × 10⁷ HeLa FRT cells were seeded on day 1, 96 h before collection, pre-synchronised twice with 2.5 mM thymidine overnight for 16 h on day 2 and day 3, then released on day 4 into media containing 330 nM nocodazole for 16 h overnight before collection. The cells were transfected with Bub1 constructs between thymidine blocks on day 3 using lipofectamine 2000 (Life Technologies). The mitotic population was collected by shake-off, washed once in ice-cold PBS and cell pellets lysed on ice for 45 min in 500 μl lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.1% NP-40, supplemented with phosphatase inhibitor and protease inhibitor cocktails (both Roche)) followed by passage 10 times through a needle. Lysate was cleared by centrifugation at 21,000g and 1 mg cleared lysate added to 40 μl GFP-Trap_A beads (Chromotek) and incubated with for 2 h at 4 degrees with rotation.
Affinity purified complexes were washed three times with 500 μl complete lysis buffer and eluted with 50 μl 3 × LDS lysis buffer. Samples were analysed by western blot using the following antibodies: BubR1 (home-made, 1:1,000), Bub1 (AbCam ab54893, 1:500) and Bub3 (BD Transduction, 611731, 1:500). Uncropped versions of blots appear in Supplementary Fig 9.

Tissue protein samples were prepared in RIPA buffer (Beyotime Biotechnology, Haimen, China) containing phosphatase inhibitor and protease inhibitor cocktails (Roche Applied Science, Indianapolis, IN, USA). Samples containing 20 μg of protein were subjected to sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, and the proteins were then transferred to a polyvinylidene difluoride membrane. The membrane was blocked with Tris‐buffered saline‐Tween with 5% bovine serum albumin and incubated at 4°C overnight with primary antibodies against α‐SMA (1:1000; AF1032, Affinity Biosciences), Collagen I (1:1000; AF7001, Affinity Biosciences), TGF‐β1 (1:1000; AF1027, Affinity Biosciences), eNOS (1:1000; AF0096, Affinity Biosciences), nNOS (1:1000; AF6249, Affinity Biosciences) or β‐actin (1:1000; 20536‐1‐AP, Proteintech, China). After hybridization of secondary antibodies (1:5000 or 1:10000; ProMab, USA), the immunoreactive proteins were detected through an enhanced chemiluminescence detection system (Pierce, Thermo Fisher Scientific, Rockford, IL, USA).